Metabolism is built of complex networks of enzymes that form links by sharing substrate and product molecules. Some of these shared molecules, which are often reactive, are not freely diffusing, but rather, their motion is directed, or channeled from one enzyme to another. In general, the mechanisms by which reactive molecules are passed between enzyme active sites are poorly understood. The goal of this project is to understand how the intermediates of proline catabolism are channeled from proline dehydrogenase (PRODH) to ?1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH). PRODH is a flavoenzyme that catalyzes the oxidation of proline to P5C. P5C is a reactive molecule that forms a nonenzymatic, pH-dependent equilibrium with the reactive carbonyl species glutamic semialdehyde (GSA). P5CDH is an NAD+dependent enzyme that catalyzes the oxidation of GSA to glutamate. The intermediate P5C/GSA is common to both the proline catabolic and synthetic pathways, and to arginine biosynthesis. P5C/GSA also influences many biological processes, including apoptosis, reactive oxygen species generation and RNA translation initiation. This project will use the bacterial bifunctional enzyme, Proline utilization A (PutA), as a model to understand channeling in detail. In PutAs, PRODH and P5CDH are fused into a single, large protein. The recent crystal structure of a PutA has revealed a unique system of internal cavities and tunnels that is hypothesized to function as both a reaction chamber for the hydrolysis of P5C to GSA and a protected pathway that facilitates transport of GSA to the P5CDH active site. Steady-state and rapid reaction kinetic data reported here also support a channeling mechanism for PutA. These initial observations furthermore suggest the hypothesis that monofunctional PRODH and P5CDH enzymes, such as those found in humans, interact and engage in intermolecular channeling. Channeling in bacterial homologs of the human enzymes will also be studied. This proposal has three aims: 1. Establish the structural and kinetic framework underlying substrate channeling in PutAs. 2. Investigate the mechanism of substrate channeling in PutA. 3. Explore substrate channeling and protein-protein interactions in monofunctional PRODH and P5CDH. Completion of these aims will provide a comprehensive, yet detailed, understanding of substrate channeling in proline catabolism. PUBLIC HEALTH RELEVANCE: This project proposes detailed biochemical and structural studies of the enzymes that recycle the amino acid proline by oxidizing it to glutamate. Genetic defects in these enzymes lead to hyperprolinemia disorders, which can be associated with mental retardation, higher frequency of febrile seizures and increased susceptibility to the disabling brain disorder schizophrenia. Also, one of the enzymes, proline dehydrogenase, helps reduce carcinogenesis in humans by serving as a reactive oxygen species generator in the cell death cascade mediated by tumor suppressor p53. The proposed research will examine how reactive intermediates are passed between proline recycling enzymes in a process known as substrate channeling. It is proposed that channeling is a fundamental aspect of the proline oxidation process.